Introduction Glioblastoma multiforme(GBM),a malignant brain tumor,is highly invasive and use brain microvessels to migrate and invade.Studying the perivascular invasion/migration of GBM may enable new possibilities in...Introduction Glioblastoma multiforme(GBM),a malignant brain tumor,is highly invasive and use brain microvessels to migrate and invade.Studying the perivascular invasion/migration of GBM may enable new possibilities in GBM therapy.However,the lack proper 3D study models that recapitulate GBM hallmarks restricts investigating cell-cell/cell-molecular interactions in tumor microenvironments.In this study,we created GBM-vascular niche models through 3D bioprinting [1-2] using patient-derived GBM cells with sternness(GSC:glioblastoma stem cells),vasculature endothelial cells(ECs),mural cells,and various hydrogels.Materials and methods Three GBM-vascular models were designed:Model A with large vessels and GBM spheroid;Model B with large-and micro-vessels,and GBM spheroid;Model C with large-and micro-vessels and scattered GBM cells.Large channels were created by sacrificial bioprinting.Microvessel network was formed through self-assembly of ECs(HUVEC or brain EC)and mural cells(fibroblast,pericytes,and/or astrocytes).Three GBM cell types were used in the study:SD02 and SD03 are GSCs;U87MG is a commercially-available GBM cell line.Collagen type I or fibrin hydrogel have been used as major scaffold materials.For drug treatment,Temozolomide in culture medium was perfused through large vasculatures in Model A.Results and discussion Three different GBM-vascular models were successfully fabricated and culture for 2-10.GSCs cultured in these models maintained sternness and heterogeneity during the long-term cultures.In Model A,GSCs actively invaded into the surrounding tissues(~Day26),initially regressed in response to the drug(~Day50),then developed therapeutic resistance and resumed aggressive invasion(~Day57).In Model B and C,three GBM types presented distinctive invasion patterns and EC-interactions.SD02 cells showed a spiky invasion pattern with elongated morphology.SD03 cells showed a more dispersed invasion pattern with many single cell migrations towards surrounding microvessels.U87MG cells showed a blunt invasion pattern,caused EC death in the spheroid form;however,the EC death was significantly reduced in the scattered single cell form.Conclusions In this study,we have created GBM-vascular niche models that can recapitulate various GBM characteristics such as cancer sternness,tumor type-specific invasion patterns,and drug responses with therapeutic resistance.Our models have a great potential in investigating patient-specific tumor behaviors under chemo-/radio-therapy conditions and consequentially helping to tailor personalized treatment strategy.The model platform is capable of modifying multiples variables including ECMs,cell types,vascular structures,and dynamic culture condition.Thus,it can be adapted to other biological systems and serve as a valuable tool for generating customized microenvironments.展开更多
目的:在体外观察血管内皮生长因子(VEGF)单克隆抗体对C6胶质瘤细胞增殖、侵袭和迁移力的影响。方法:采用氯化钴(Co Cl2)模拟缺氧环境,予以0、0.1、1、10μg/m L的VEGF抗体,对常氧或缺氧条件下对C6胶质瘤细胞进行处理。通过MTT实验检测...目的:在体外观察血管内皮生长因子(VEGF)单克隆抗体对C6胶质瘤细胞增殖、侵袭和迁移力的影响。方法:采用氯化钴(Co Cl2)模拟缺氧环境,予以0、0.1、1、10μg/m L的VEGF抗体,对常氧或缺氧条件下对C6胶质瘤细胞进行处理。通过MTT实验检测细胞增殖,Transwell实验检测细胞的侵袭和迁移能力,Western blotting检测HIF-1α蛋白、FAK/Pyk2总蛋白及磷酸化FAK-Tyr397/Pyk2-Tyr402表达水平变化。结果:200μmol/L Co Cl2明显抑制C6细胞增殖(P<0.05),10μg/m L VEGF抗体可明显抑制C6细胞增殖(P<0.05)。与常氧相比,缺氧可使C6细胞的迁移、侵袭能力增强(P<0.05)。常氧和缺氧下,VEGF抗体的干预均增强C6细胞的迁移、侵袭能力(P<0.05),且相对于常氧,缺氧可进一步增强VEGF抗体促C6细胞迁移、侵袭的作用(P<0.05)。Co Cl2干预可增加C6细胞中HIF-1α的含量;在常氧和缺氧情况下,只有1μg/m L VEGF抗体可降低FAK的总蛋白量(P<0.05);在缺氧情况下,VEGF抗体可明显增高Pyk2-Tyr402位点的磷酸化水平。结论:在缺氧情况下,VEGF抗体的干预可使Pyk2-Tyr402位点的磷酸化水平增高,引起C6细胞的侵袭迁移能力进一步增强。展开更多
基金supported mainly by grants from American Heart Association Scientist Development Grant ( 12SDG12050083 to G.D.)National Institute of Health ( R21HL102773,R21HD090680,R01HL118245 to G.D.)National Science Foundation ( CBET-1263455,CBET-1350240 to G.D.)
文摘Introduction Glioblastoma multiforme(GBM),a malignant brain tumor,is highly invasive and use brain microvessels to migrate and invade.Studying the perivascular invasion/migration of GBM may enable new possibilities in GBM therapy.However,the lack proper 3D study models that recapitulate GBM hallmarks restricts investigating cell-cell/cell-molecular interactions in tumor microenvironments.In this study,we created GBM-vascular niche models through 3D bioprinting [1-2] using patient-derived GBM cells with sternness(GSC:glioblastoma stem cells),vasculature endothelial cells(ECs),mural cells,and various hydrogels.Materials and methods Three GBM-vascular models were designed:Model A with large vessels and GBM spheroid;Model B with large-and micro-vessels,and GBM spheroid;Model C with large-and micro-vessels and scattered GBM cells.Large channels were created by sacrificial bioprinting.Microvessel network was formed through self-assembly of ECs(HUVEC or brain EC)and mural cells(fibroblast,pericytes,and/or astrocytes).Three GBM cell types were used in the study:SD02 and SD03 are GSCs;U87MG is a commercially-available GBM cell line.Collagen type I or fibrin hydrogel have been used as major scaffold materials.For drug treatment,Temozolomide in culture medium was perfused through large vasculatures in Model A.Results and discussion Three different GBM-vascular models were successfully fabricated and culture for 2-10.GSCs cultured in these models maintained sternness and heterogeneity during the long-term cultures.In Model A,GSCs actively invaded into the surrounding tissues(~Day26),initially regressed in response to the drug(~Day50),then developed therapeutic resistance and resumed aggressive invasion(~Day57).In Model B and C,three GBM types presented distinctive invasion patterns and EC-interactions.SD02 cells showed a spiky invasion pattern with elongated morphology.SD03 cells showed a more dispersed invasion pattern with many single cell migrations towards surrounding microvessels.U87MG cells showed a blunt invasion pattern,caused EC death in the spheroid form;however,the EC death was significantly reduced in the scattered single cell form.Conclusions In this study,we have created GBM-vascular niche models that can recapitulate various GBM characteristics such as cancer sternness,tumor type-specific invasion patterns,and drug responses with therapeutic resistance.Our models have a great potential in investigating patient-specific tumor behaviors under chemo-/radio-therapy conditions and consequentially helping to tailor personalized treatment strategy.The model platform is capable of modifying multiples variables including ECMs,cell types,vascular structures,and dynamic culture condition.Thus,it can be adapted to other biological systems and serve as a valuable tool for generating customized microenvironments.
文摘目的:在体外观察血管内皮生长因子(VEGF)单克隆抗体对C6胶质瘤细胞增殖、侵袭和迁移力的影响。方法:采用氯化钴(Co Cl2)模拟缺氧环境,予以0、0.1、1、10μg/m L的VEGF抗体,对常氧或缺氧条件下对C6胶质瘤细胞进行处理。通过MTT实验检测细胞增殖,Transwell实验检测细胞的侵袭和迁移能力,Western blotting检测HIF-1α蛋白、FAK/Pyk2总蛋白及磷酸化FAK-Tyr397/Pyk2-Tyr402表达水平变化。结果:200μmol/L Co Cl2明显抑制C6细胞增殖(P<0.05),10μg/m L VEGF抗体可明显抑制C6细胞增殖(P<0.05)。与常氧相比,缺氧可使C6细胞的迁移、侵袭能力增强(P<0.05)。常氧和缺氧下,VEGF抗体的干预均增强C6细胞的迁移、侵袭能力(P<0.05),且相对于常氧,缺氧可进一步增强VEGF抗体促C6细胞迁移、侵袭的作用(P<0.05)。Co Cl2干预可增加C6细胞中HIF-1α的含量;在常氧和缺氧情况下,只有1μg/m L VEGF抗体可降低FAK的总蛋白量(P<0.05);在缺氧情况下,VEGF抗体可明显增高Pyk2-Tyr402位点的磷酸化水平。结论:在缺氧情况下,VEGF抗体的干预可使Pyk2-Tyr402位点的磷酸化水平增高,引起C6细胞的侵袭迁移能力进一步增强。
